The major goal of the work proposed in this application is the investigation of the structure-function relationships in E. coli RNA polymerase holoenzyme by a genetic, physiological and biochemical analysis of mutants. In addition, second site revertants of certain mutant classes will be selected with the goal of identifying previously unknown transcriptional factors. The research is organized into two project areas. A. What is the in vivo role of the sigma subunit of RNA polymerase in selective initiation? Cellular regulation of both synthesis and degradation of sigma is being investigated by a variety of techniques. In addition, the role of different regions of the sigma polypeptide in selective initiation is being studied. Mutants altered in recognition of promoters or positive regulatory factors are being made and analyzed genetically, physiologically and biochemically. B. What is the structural and functional organization of the beta and beta prime subunits of core RNA polymerase? Structure-function relationships for 5 different classes of RNA polymerase mutants: rifampicin resistant mutants, streptolydigin resistant mutants, mutants with altered sigma interaction, mutants with altered DNA binding and mutants that exhibit a dominant lethal phenotype are being studied. Deletion mapping will be used to locate precisely the mutations within rpoB (coding for Beta) or rpoC (coding for Beta'). In vivo analysis of regulation of gene activity and in vitro analysis of DNA binding and transcription will enable assessment of the nature of the functional alteration in the mutant RNA polymerase. Based on such studies, we will be able to determine whether mutations resulting in the same or similar functional alterations are clustered on the genetic map. DNA sequencing of clustered mutations will enable us to correlate functional defects with changes in the primary and secondary structure of the protein. Finally, second site revertants of some mutant classes will be used to identify factors interacting with polymerase. The subunit structure of RNA polymerase is conserved throughout the bacterial world. Thus, it is likely that our conclusions about structural organization will be applicable to other procaryotes including those which are important either as disease-causing organisms or as model systems for the analysis of development.